Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications

Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity

Background: Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown.Objective: Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.Methods: Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.Results: HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p2 = 0.35, p = 0.043).Conclusion: Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.</p

KTN (RCK) Domains Regulate K+ Channels and Transporters by Controlling the Dimer-Hinge Conformation

KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K+ channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membrane-integrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K+ efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux

Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity

<div><p>Background</p><p>Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown.</p><p>Objective</p><p>Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.</p><p>Methods</p><p>Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.</p><p>Results</p><p>HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r² = 0.35, p = 0.043).</p><p>Conclusion</p><p>Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.</p></div

Evidence That Two Enzyme-derived Histidine Ligands Are Sufficient for Iron Binding and Catalysis by Factor Inhibiting HIF (FIH)*S⃞

A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1α786–826 peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1α786–826/788–806 implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron

ΔCt Bacteroidetes: ΔCt Firmicutes Ratio Correlates with Exercise Distance.

<p>There was a significant, but modest inverse relationship between the ΔCt Bacteroidetes: ΔCt Firmicutes ratio and the distance recorded for the combined LF/Ex and HF/Ex mice. Data analyzed by Pearson product-moment correlation coefficient with an alpha level of p<0.05. n = 6 all groups.</p

Beta Diversity is Elevated by HF Diet and Exercise.

<p>Significant differences indicated as follows: “*” p<0.05 for diet effect, “†” p<0.05 activity effect and “‡” p<0.05 diet and activity interaction. n = 6 mice/group.</p

Clustering of Samples Based on Litter, Diet and Activity.

<p>Principal coordinate analysis (PCA) was performed based on the weighted UniFrac distance matrix generated from sequencing fecal 16S rRNA gene in samples from mice at week 0 and 12 of the diet and activity protocol. A. Clustering demonstrated by litter at week 0. B. No clustering demonstrated by litter at week 12. C. No clustering demonstrated by diet and activity at week 0. D. Clustering demonstrated by diet and activity at week 12. The top panels show the PCA keyed by litter (6 liters, 1–6, of 4 mice each) and the bottom panels show the PCA keyed by diet and activity group. The X-axis represents the primary coordinate, the Y-axis represents the secondary coordinate. Axis numbering represents the relative distance between samples based on the weighted UniFrac distance matrix.</p

Diet and Activity Altered the Relative Level of Bacteroidetes and Firmicutes.

<p>A. The fold change in Bacteroidetes and Firmicutes was determined from the 2^−ΔΔCt values calculated from the ΔCt values generated by quantitative polymerase chain reaction (qPCR) using primers specific to each phyla (one-way ANOVA). B. Criterion validity of qPCR was examined by correlating the ΔCt-Bacteroidetes: ΔCt-Firmicutes ratio with the %-Bacteroidetes: %- Firmicutes ratios from sequencing. Data was analyzed by Pearson product-moment correlation coefficient and alpha level of p<0.05.</p

Phylum Level Changes with Diet and Activity.

<p>At week 12, diet and activity changed the levels of two major and two minor phyla of bacteria. Data were analyzed by 2-way ANOVA with a Sidak post hoc test. Significant differences indicated as follows: “*” p<0.05 for diet effect, “†” p<0.05 activity effect and “‡” p<0.05 diet and activity interaction. n = 6 mice/group.</p